The goal of this proposal is to apply RNA interference (RNAi) technology to gain a better understanding of the p53 pathway, and its role in programmed cell death (apoptosis), immortalization and genomic instability. The p53 pathway comprises the most mutated genes in cancer. Although p53 has been the focus of a tremendous research effort, much still remains unknown about p53 and the specific pathways that mediate its tumor-suppressive activities. For example, the mechanism by which specific pathways within the p53 network are activated is not yet well understood. My recent data suggest that at very low levels of p53 ('residual' p53), some p53-mediated phenotypes are activated while others are not. Those data establish that residual p53 is sufficient to protect the genome from instability, but does not prevent immortalization. The ability to de-couple these two p53-dependent pathways is novel and allows for a more precise dissection of the p53 network. In this proposal, I will utilize RNAi technology in primary mouse embryo fibroblasts (MEFs) to induce the residual p53 phenotype and explore mechanisms underlying dose-dependent p53 activities. Moreover, I will utilize a short hairpin RNAi library to identify genes involved in the p53-dependent immortalization of MEFs. Consequently, I hope to identify a profile of p53-regulated genes that are sensitive to discrete levels of cellular p53 in normal cells. I expect these studies will provide new information that will facilitate the rational design of anti-cancer agents targeting the p53 pathway. [unreadable] [unreadable] [unreadable]